The Clostridial neurotoxins are the most potent toxins known to man. When Clostridium botulinum bacteria are ingested orally they produce botulinum toxin (BT). BT is absorbed from the gastrointestinal tract and is transported by the circulatory system to muscles throughout the body. The BT binds to and blocks neuromuscular transmission from motor neurons causing a fatal paralysis known as botulism.
An unusual attribute of the BT is that its action lasts for months but the patient completely recovers. As a result of this unique attribute, BT has many clinical uses. At present, the local injection of small doses of BT is used to decrease or block muscle activity in a wide variety of clinical motor disorders. More recently, the use of BT has been extended to block autonomic nerves that use the same neurotransmitter used in neuromuscular transmission, namely, acetylcholine.
The other class of Clostridial bacteria is Clostridia tetani. These bacteria infect wounds and produce tetanus toxin. Tetanus toxin (TT) is released from the site of the infection and is distributed by the circulatory system to motor neurons throughout the body. Instead of acting on the motor neurons directly, the tetanus toxin is transported to the central nervous system where it blocks neurons that normally inhibit motor neuron activity. The result is a gradually increasing tone in affected muscles that culminates in a widespread spasm of muscles throughout the body. The resulting spastic paralysis is often fatal with death resulting from respiratory depression or circulatory collapse.
Tetanus has been recognized as a disorder since antiquity and it is still common throughout the world. Many countries routinely vaccinate children with tetanus toxoid, an attenuated form of the toxin that is exposed to formaldehyde to remove its biological activity while retaining its antigenicity. Tetanus toxoid is the largest biologic product in the pharmaceutical industry.
The action of the tetanus toxin lasts from weeks to months. Once TT enters into and blocks neurotransmission from neuron synapses the process is irreversible. Recovery of function requires the growth of a new process from the neuron that eventually reconnects to the motor neuron and restores the inhibitory activity back to normal levels. The time required for this recovery varies from weeks to up to five months (Struppler, A., et al. Arch Neurol, 8, 162-1782, (1963)).
The extremely broad range of TT actions allows it to either excite or inhibit practically any part of the nervous system for prolonged periods of time with a single injection. Since the nervous system closely monitors and controls nearly every organ and physiological function it has been unexpectedly found that TT can have extensive beneficial utility for the treatment or amelioration of a wide variety of clinical disorders.
Tetanus is a systemic intoxication by the tetanus toxin which is characterized by progressive spastic contraction of the skeletal muscles and overactivity of the autonomic nervous system that is often fatal.
Other than the systemic disorder three lesser known variants or tetanus are known. These are neonatal, cephalic and local tetanus. Neonatal tetanus is a fatal intoxication of newborn babies that is manifest as a systemic flaccid paralysis. Cephalic tetanus occurs on the face and combines a localized paralysis, most often of the facial nerve, with a surrounding area of muscle spasm (Dastur, F. D., et al., Journal Of Neurology, Neurosurgery And Psychiatry 40(8), 782-6 (1977)). Local tetanus is an isolated spasm of a muscle group or limb that may progress to systemic tetanus or resolve over weeks to months (Johns Hopkins Medical Journal, 149(2) 84-8, (1981); Jain, S., et al., Journal Of Neurology, 228(4), 289-93, (1982)).
The tetanus toxin has some unique properties that have made it perhaps the most studied of all biological toxins. For example, the tetanus toxin binds to all types of neurons. Although its primary affinity is to bind to motor neurons, TT also binds to neurons of the autonomic nervous system and sensory neurons (Stockel, K., et al., Brain Research, 99, 1-16 (1975)). In contrast, the botulinum toxins principally bind to motor neurons.
Tetanus toxin requires multiple specific steps to cause its effects in neurons. These steps include (i) peripheral binding; (ii) internalization; (iii) retrograde transport; (iv) central binding; and (v) transmembrane internalization.
In peripheral binding the toxin binds to the surface of the cell. TT binds to the presynaptic membrane of practically all neurons. In addition it also binds to the membrane of the neuron's axon. The receptors to which TT binds are a class of molecules known as gangliosides. BT also binds to gangliosides, however BT appears to bind principally to those on the presynaptic membrane of cholinergic neurons, whereas TT binds to the pre-synaptic membrane of most if not all neurons.
In the internalization step the toxin is brought into the cell. TT is brought into the neuron by the process of forming a vesicle. While TT remains inside the vesicle, although physically inside the neuron, the toxin is separated from the cytoplasm of the neuron by a membrane. In contrast, BT is thought to require a second molecule on the presynaptic membrane to bind to before being internalized. After BT binds to the second molecule it passes through the cell membrane directly into the cytoplasm, which is why it exerts its effect at the peripheral presynaptic membrane.
During the retrograde transport the vesicles containing TT are transported to the cell body in the central nervous system. The vesicle then fuses with the cell membrane of the cell body or its dendrites thereby depositing TT into the extracellular space between the motor neuron on the processes of other neurons synapsing onto the motor neuron (Hilbig, G., K. O. Räker, et al., Naunyn-Schmiedebergs Archives Of Pharmacology, 307(3), 287-90, 1979.
In the central binding step TT can bind to all neurons. However, TT has a much greater affinity for the inhibitory neurons. At low concentrations, tetanus has greater affinity for the neurons that use the inhibitory neurotransmitters GABA and glycine (Montecucco, C. et al., Q Rev Biophys, 28(4), 423-72, (1995)). At higher concentrations, it blocks all neurotransmitters. Finally, tetanus toxin has a local effect on axons that causes a local block of the propagation of action potentials. The mechanism for this is unknown but the result is similar to the action of a local anesthetic.
During the transmembrane internalization, once the toxin binds to a second neuron it is internalized and produces its toxic effect.
The primary mechanism of action of TT is to block the release of vesicles from a cell. In neurons these vesicles contain neurotransmitters. The proteins that are involved in the attachment of a vesicle to the inner membrane of a cell are the SNARE (synaptosome associate protein receptor) family of proteins. These proteins are part of the mechanism by which intracellular vesicles dock to cell membranes and release their contents. Specifically, tetanus toxin cleaves VAMP (vesicle associated membrane protein). Botulinum toxins A and E cIcavc SNAP-25; and botulinum toxin C cleaves SNAP-25 and syntaxin; tetanus toxin and botulinum neurotoxins type B, D, F and G cleave VAMP, an integral protein of the neurotransmitter containing synaptic vesicles.
The mechanism of vesicle release is common to all cells from yeast to the cells of humans. The TT molecule is composed of a heavy chain that is responsible for its specific binding and transport properties, and a light chain that actually performs the catalytic action on the VAMP protein. There are a few non-neuronal cells in which TT is capable of entering and performing its action and these will be discussed in the examples.
Multiple experiments have shown that even if TT is incapable of binding and entering into a type of cell it can be inserted by a variety of mechanisms. Once inside the cell TT cleaves VAMP and disables vesicle release. Different cells use the mechanism to secrete hormones, neuropeptides, lysozyme proteins, and other substances. Whatever specific substance is secreted by the cell it will be blocked by TT if secretion requires the use of VAMP protein.
Cells can be made susceptible to TT by placing gangliosides onto the external surface of the cell membrane. Another manner of inserting TT into cells is by chemically combining the TT, or at a minimum its light chain, with a second molecule that is capable of binding to the cell. In addition the TT can be inserted by micro injection using micropipettes, pressure injection, by incorporation into lysosomes, or by temporarily making the cell membrane permeable to TT.
Motor neurons are the primary target of TT toxicity. Motor neurons refer to cholinergic neurons that innervate the large extrafusal muscle fibers of skeletal muscle. Within skeletal muscle are smaller intrafusal muscle fibers and these are innervated by a smaller cholinergic motor neuron called a gamma neuron. These also are intoxicated by TT. Finally, smooth muscle is also innervated by cholinergic neurons. Although the vast majority of these neurons are cholinergic and are formally considered part of the autonomic nervous system they are sometimes grouped with the other motor neurons for discussion because they have certain biologically similar properties. TT has been shown to bind to and intoxicate the motor neurons of smooth muscles in the same manner as it does to striated muscle fibers.
It should be noted that at high doses TT can cause a flaccid paralysis by blocking motor neuron activity both in the periphery, at the neuromuscular junction, and centrally, by blocking all afferent input from both excitatory and inhibitory axons. Which of the two areas predominate in a given case of intoxication is dependent on where the TT infection is and how it progresses.
In cephalic tetanus all three actions of TT can occur together. At the site of infection the muscle exhibits a flaccid paralysis as TT levels are high and the neuromuscular synapses are blocked directly. In addition high levels of the toxin are transported back to the brainstem to block central nervous system input. However at variable distances surrounding the site of infection the concentration of TT falls until areas are seen in which muscles are in spasm (Dastur, F. D., et al., Journal Of Neurology, Neurosurgery And Psychiatry 40(8), 782-6 (1977)).
Tetanus toxin can be measured by weight or, more commonly, by biological assay. The effective dose of tetanus is measured in units, the amount of tetanus toxin that is lethal to 50% of mice when injected subcutaneously. A unit of tetanus toxin may range between 0.1 to 100 ng of toxin per kg of mouse body weight. In the mouse hemi diaphragm assay, a 500 times higher dose of tetanus toxin is needed to cause a flaccid paralysis (Bigalke, H. et al., Naunyn Schmiedebergs Arch Pharmacol, 312(3), 255-63, (1980)). Therefore at equivalent weights tetanus toxin would be expected to cause a spastic paralysis while botulinum toxin causes a flaccid paralysis.
Tetanus toxin has the same effects on autonomic neurons as it does on motor neurons (Abboud, F. M., Hypertension, 4 (3 Pt 2), 208-25, (1982)). In systemic tetanus excitation of the autonomic nervous system is prominent and manifest by such symptoms as high blood pressure (Toriya, Y., T. et al. Endodontics And Dental Traumatology, 13 (1), 6-12, (1997)), erratic changes in blood pressure, high fever, and profuse sweating. Therefore, low concentrations result in increased autonomic tone with physiological changes resulting in all organs affected.
The autonomic nervous system is divided into the parasympathetic system which uses acetylcholine as its neurotransmitter and the sympathetic systems which uses epinephrine as the neurotransmitter. In both the parasympathetic and sympathetic systems neurons do not reach the entire distance from the central nervous system to the peripheral organ. Instead the distance requires two neurons with a synapse somewhere in the periphery. In the sympathetic system these are grouped together in a limited number of large ganglia located near the spinal column. In the parasympathetic organ the synapse is usually located in a smaller ganglia near the target organ. In both systems the neurotransmitter used in the ganglia synapses is always acetylcholine.
Although the peripheral neurons of the sympathetic nervous system may all use norepinephrine as their neurotransmitter, the response of the target organ cells is dependent on the type of receptor. There are three types of adrenergic receptors: alpha (smooth muscle contraction), beta1 (cardiac acceleration and fatty acid mobilization) and beta 2 (smooth muscle relaxation). Note that the exact effect of norepinephrine may be entirely opposite on different muscles based on the type of receptors found on the surface. Much of this information is known for most organs that are clinically relevant.
The presence of ganglia and multiple neurotransmitters increases the complexity of the autonomic system relative to the motor system. An organ is usually innervated by both parasympathetic and sympathetic neurons and they usually have opposite actions on the target. Therefore an understanding of the anatomy and physiology of the target organ is important in planning the location of a TT injection so that the desired effect is to be achieved.
Sensory neurons can also be blocked by tetanus toxin. Sensory neuropathies are part of the symptom complex of clinical tetanus. One aspect of Clostridial tetanus infection is that pain and inflammation at the site of infection are much lower then would be expected in such serious infections. Therefore, it is believed that T′ blocks sensory neurons.
In addition to neurotransmitters, which are usually used to directly communicate with other neurons through synaptic connections, neurons release neuropeptides from motor, sensory and autonomic nerves (SP, substance P; NKA, neurokinin A; CGRP, calcitonin gene-related peptide; NPY, neuropeptide Y, interleukins and growth factors). These neuropeptides have many different effects but one of the most important is vasodilatation and inflammation. (Bigalke, H. et al, Naunyn Schmiedebergs Arch Pharmacol, 312(3), 255-63, (1980)). These neuropeptides are released by the same vesicle mechanism as neurotransmitters and therefore can be blocked by TT.
The SNARE proteins and the vesicle release mechanism are used by cells for purposes other then the release of neurotransmitters. In fact, the release of practically all cellular secretions depends on this mechanism. These include the release of hormones, enzymes, and inflammatory modulators, mucus secretions from respiratory, digestive and urinary glands, and inflammatory modulators from nerves and white blood cells (Alexander, E. A. et al., American Journal of Physiology, 273 (6 Pt 2), F1054-7 (1997)). Cells known to internalize tetanus toxin include macrophages, endocrine cells, and renal cells (Huet de la Tour. E., et al., Journal Of The Neurological Sciences, 40(2-3), 123-31, (1979)).
In addition to their effect on SNARE proteins, Clostridial toxins have been shown to interfere with other cell activities. For example, it can prevent actin molecules from forming into filaments. Actin is the main cellular skeleton protein involved in cell shape and movement. This action can block the contraction of muscle cells as well as stop the migration of white blood cells and possibly malignant cells also. The toxins also interfere with cell signaling. Specifically, receptors on a cell's surface respond to specific molecules by promoting a cascade of secondary proteins that in turn result in a variety of cell functions from changes in morphology to secretion.
In “Ophthalmic and Reconstructive Surgery,” 16 (2), 101-13, (2000), Fezza J. P. et al. disclose the use of tetanus toxin to cause localized orbiculari oculi weakness without producing systemic tetany in immunized rabbits. Potential uses of tetanus toxin in treatment of blepharospasm and hemifacial spasm are suggested without provisions of any detailed information regarding dosage or other description useful to one skilled in the art seeking to use the tetanus toxin to treat these conditions.
U.S. Pat. No. 5,989,545 to Foster et al. describes the use of the light chain of a clostridial neurotoxin by itself or linked to other moieties as a pharmaceutical for the treatment of pain. Foster et al. do not disclose the use of the entire molecule of tetanus toxin.
U.S. Pat. No. 5,714,468 to Binder describes the use of a fragment of tetanus toxin to reduce pain in migraine headaches. U.S. Pat. No. 5,670,484 to Binder discloses a method for treatment of cutaneous cell-proliferative disorders with Botulinum toxin A and tetanus toxin. In both patents, Binder uses the same TT dosages as are used for BT. Moreover, he discourages the use of TT for beneficial purposes because be found that TT to be too toxic at the dosages disclosed in his patents.
U.S. Pat. No. 5,766,605 to Sanders et al. describes the control of autonomic nerve function in a mammal by administering to the mammal a therapeutically effective amount of Botulinum toxin. There is no disclosure of tetanus toxin.
Despite the apparent effects of neurotoxins on motor, autonomic and sensory neurons, the use of such toxins, and especially tetanus toxin in animals, including humans, has been limited and has never been used for clinical applications. Thus, there remains a need in the medical art for methods of treating patients with tetanus toxin that can cause an increase or decrease in neural activity at selected sites of the patient. Similarly, there is still a need in the medical arts for methods using tetanus toxin to treat clinical disorders caused by improper cellular activity, such as inflammatory conditions. Further, there remains a need for pharmaceutical formulations that can be delivered to a patient to achieve clinically beneficial results or treat certain dysfunctions, while eliminating or minimizing dependence, tolerance, and side effects associated with more conventional drugs.